Immunosuppressant drugs such as sirolimus (also known as rapamycin), tacrolimus, everolimus, temsorolimus and cyclosporine are effective for the treatment of organ or tissue rejection following transplant surgery, of graft versus host disease and of autoimmune diseases in humans. During immunosuppressant drug therapy, monitoring the blood concentration levels of the immunosuppressant is an important aspect of clinical care because insufficient drug levels lead to graft (organ or tissue) rejection and excessive levels lead to undesired side effects and toxicities. Blood levels of immunosuppressant are therefore measured so drug dosages can be adjusted to maintain the drug level at the appropriate concentration. Diagnostic assays for determination of immunosuppressant blood levels have thus found wide clinical use.
Initially, the immunosuppressant must be extracted and separated from the other components of the patient sample. The bulk of the immunosuppressant drug in the patient sample is present in a complex with various “carrier” molecules, such as binding proteins. Sirolimus, tacrolimus and cyclosporine are found predominately in the red blood cells of patient specimens and are associated with specific binding proteins, FKBP for sirolimus and tacrolimus, and cyclophilin for cyclosporine. An accurate measurement of the total drug concentration in the specimen requires that the drug bound to the binding proteins be liberated prior to quantitation. Following its extraction from the binding proteins, the drug can be measured in a number of different ways, including by immunoassay or chromatography with absorbance or mass spectrophotometric detection.
Extraction of sirolimus from its binding proteins in blood is often accomplished by treatment with organic solvents, such as, acetonitrile, methanol, or diethyl ether. These solvents denature the binding proteins and liberate the drug. The use of organic solvents can be problematic, however, when an immunoassay is subsequently used to detect the liberated drug because most organic solvents that will quickly and completely denature the binding proteins are not compatible with immunoassays. They are either too harsh or they create a biphasic sample. Methanol has typically been employed to extract sirolimus, tacrolimus or cyclosporine from blood specimens before immunoassay. However, a careful balance must be achieved such that the methanol concentration is sufficient to liberate the drug from the binding protein, but not so great as to interfere with the subsequent immunoassay. The use of methanol and other typically used organic solvents create an additional problem because these solvents have higher vapor pressure than water. As a result, the extraction supernatant containing the immunosuppressant drug evaporates quickly which causes inaccuracy in the measurement of the drug concentration. The widely used methanol or acetonitrile solvents also create handling and disposal issues for the laboratories.
Immunoassays for immunosuppressant drugs are available in a variety of formats, but all use the binding of an antibody or binding protein (e.g. FKBP) to the immunosuppressant drug. A commonly used prior art immunoassay is an assay which involves the binding of a first antibody to the immunosuppressant and the binding of labeled immunosuppressant (e.g. acridinium-sirolimus) to the remaining free antibody binding sites, followed by quantitation by detection of the label. The effectiveness of these immunoassays is affected by the particular extraction and denaturating solvent for the immunosuppressant that is used.
It is an object of the invention to provide for use with immunoassays an improved immunosuppressant drug extraction reagent composition that has a low vapor pressure, miscibility with water, sufficient immunosupressant denaturing power and compatibility with immunoassay reagents. Such an extraction reagent composition would be advantageous as well for non-immunoassay methods (e.g. chromatographic determinations) because the lower vapor pressure, sufficient denaturing power and water miscibility would make these methods easier to use.